Dihydrofolate reductase is the site of action of several important drugs widely used in the chemotherapeutic treatment of numerous malignancies, certain bacterial infections, psoriasis, arthritis and other human diseases. Understanding the detailed mechanism of action of this enzyme and its inhibition by these drugs as well as its unique molecular biology should facilitate a rational approach to drug design and chemotherapy. Thus we continue our studies on the isolation and characterization of hepatic dihydrofolate reductase (DHFR) from various animals, i.e., chicken, beef, pork, sheep, with the rationalization that the comparative investigation of a seemingly unique feature exhibited by an enzyme from a particular animal species might lead to an overall synthesis of understanding of the general nature of this unusual enzyme. For example, the stepwise glutamylation of methotrexate decreased the inhibition of sheep liver DHFR until the polyglutamate derivative with six residues was 1/3 as effective as the original drug. On the other hand, if the hexaglutamate derivative of dihydrofolate is used as substrate, the corresponding hexaglutamate of MTX is now 2-5 fold more active than the monoglutamate. Thus the Glu chain length of both the inhibitor as well as the substrate could be important in determining DHFR inhibition in vivo. Recent speculation that the dietary intake of carotenoids may be a factor in the prevention of human cancer has stimulated a renewed interest in carotenoid nutrition and metabolism. As part of a study to obtain more detailed information on carotenoid metabolims in normal humans, we have developed a rapid procedure for the analysis of plasma carotenoids. This HPLC procedure revealed leutin and zeaxanthin, cryptoxanthin, lycopene, Alpha- and Beta-carotene as major compounds plus two unidentified peaks. The method has been used in measuring the plasma response of individuals to a standard dose of oral carotene and following long term changes in plasma carotenoid patterns.